Use of thymulin-like peptides for making pain-relieving medicines

ABSTRACT

The present invention relates to the utilization of peptide analogues of thymulin that are inactive relative to the immune system, not comprising zinc and having anti-pain activity, for manufacturing a medicine for the treatment of pain.

FIELD OF THE INVENTION

The invention relates to the utilization of peptide analogues ofthymulin for use in manufacturing anti-pain medicines.

BACKGROUND OF THE INVENTION

The majority of acute or chronic pain results from an inflammatoryreaction. The recommended treatments for reducing pain frequentlyconsist of initially reducing the inflammatory reaction.

At the present time there are two main classes of anti-inflammatorymedicines:

-   -   the non-steroidal anti-inflammatory agents (NSAIDs); and    -   the corticosteroids.

The NSAIDs and the corticosteroids have the drawback of combining anunpleasant side effect with their beneficial therapeutic effect(reduction of inflammation and pain).

In fact, the NSAIDs provoke the formation of ulcers while thecorticosteroids have an immunosuppressive action.

The ideal analgesic anti-inflammatory medicine would be a medicine nothaving side effects nor an effect on physiology nor on the immunesystem.

In addition, there is a second type of pain that is not caused byinflammation. This neurogenic pain is, furthermore, characterized by itsbeing refractory to traditional treatment, including opiates. Differenttreatment have been implemented such as the use of anti-inflammatories,anti-epileptics, anti-depressants, sympatholytic drugs or combinationsof these.

However, neurogenic pain is very protean and consequently is verydifficult to treat.

Because of these considerations, the medicines available today fortreating pain are limited in number and are sometimes ineffective. Thisineffectiveness can also be the result of an acquired tolerance to theproduct. So, the practitioner is obliged to modify his prescription. Inorder for this to be efficacious, there has to be another class ofmedicines available to him.

This explains the importance of the research in this field.

DESCRIPTION OF THE INVENTION

The peptide analogs of thymulin according to the present invention havealready been described in the patents and certificate of addition FR7715963, FR 7811870 and EP 0041019 as relates to medicines for thetreatment of auto-immune diseases, stimulation of T cells and theprevention of graft rejection. The properties of these peptides relativeto the immune system have been shown to be zinc-dependent. In fact, zinccontained in the peptide confers upon it a tetrahedral conformation,which corresponds to the active form of the molecule. In the absence ofzinc, the peptide analogs would no longer have any activity. Inaddition, it has consequently been shown that these properties,demonstrated by in vitro assays, have no affect on the immune system atin vivo trials. Moreover, no secondary effects have been produced. Thesepeptides are perfectly safe.

Numerous publications have shown that thymulin can, depending on thedose injected, induce or reduce hyperalgesia (Safieh-Garabedian et al.,Neuroimmunomodulation, 6:39-44, 1999). At low doses (on the order ofnanograms in the rat; that is 0.2 to 20 μg/kg), thymulin induceshyperalgesia, while at higher doses (on the order of micrograms in therat; in other words 50-100 μg/kg), it reduces hyperalgesia. Utilizationof thymulin was thus impossible, given its effect on the immune systemand this dose-dependent (or biphasic) effect, inducing or reducing pain.

Accordingly, the inventors were interested in peptide analogues ofthymulin that are inactive relative to the immune system. Although theydid not exhibit the initially expected activity, they verified theirspectrum of activity and confirmed that, against all expectations, theydid not exhibit this ambivalent dose-dependent effect, that they hadsolely an analgesic activity, without being zinc dependent, and thatfinally, they were shown to be active in vivo.

The inventors' findings thus led them to provide, using these peptideswhose safety had already otherwise been established, a new class ofanti-pain medicines making it possible to treat pain of inflammatoryand/or neurogenic origin.

The present invention relates to the utilization of peptide analogues ofthymulin (TAP), which are inactive relative to the immune system andhave analgesic activity, for manufacturing a medicine for the treatmentof pain.

“Peptide analogues of thymulin inactive relative to the immune system”for the purpose of this application are defined as peptide analogues ofthymulin that are inactive relative to the T-cell specific immuneresponse. In particular, these peptides do not form complexes withmetals such as zinc, for example. (Dardenne et al., PNAS, 79: 5370-5373,1982)

Utilization of the peptides according to the invention has the advantageof being effective against pain without inducing adverse secondaryeffects.

These peptides are otherwise efficacious at doses from 10 to 100 timeslower than those of classical analgesics. For example, in the rat, thedoses utilized are on the order of 1 μg per rat (or 5 μg/kg) versus 4mg/kg for non-steroidal anti-inflammatory drugs and 200 mg/kg forsteroidal anti-inflammatory drugs. Activity was also found at lowerdoses on the order of 50 to 200 ng/rat; that is from 0.25 to 1 μg/kg.

In particular, the peptides utilized according to the invention have thefollowing sequence:

X-Gln-Gly-Gly-Ser-Asn (SEQ ID NO: 47)

-   -   wherein X represents Ser, Lys-Ser, Ala-Lys-Ser, Glu-Ala-Lys-Ser        (SEQ ID NO: 48), Gln-Ala-Lys-Ser (SEQ ID NO: 49),        PyroGlu-Ala-Lys-Ser (SEQ ID NO: 50), as well as any derived        sequence comprising 1 or 2 modified amino acids, said possible        modifications being of the following type:

PyroGlu: D-PyroGlu, Glu, Gln

Gln: Z-Gln, D-Gln, Pro, Cys (S—CONH2), Met(O), Glu, Glu(γ-cyano),Glu(γCS—NH2), D-Glu, Asn, NorVal

Ala: D-Ala, Z-Ala, Ac-Ala

Lys: Arg, D-Lys, N-γ-Z-Lys, Lys(N6 acetyl), Orn, Har, 2-amino-hexanoyl,2,6-diamino-hexynoyl, 2,6-diamino-hexenoyl, Hep, D-Lys(N6-acetyl)

Ser: Ala, (N-methyl)Ser, D-Ser, Thr

Gly: Ala, Ser, D-Ala, D-Leu

Asn: CyanoAla, Thio-Asn, Asp, Gln, Glu, β-Ala-NH2, D-Asn, Asn-NH2

Reference to the “thymulin analogues” clearly excludes thymuline, thepeptide having the following sequence:PyroGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (SEQ ID NO: 46).

In particular, the invention envisages protection of utilization of thefollowing peptides:

 (1) PyroGlu-Ala-Lys-Ser-Gln-Gly- (SEQ ID NO: 1) Gly-Ser-Asp  (2)PyroGlu-Ala-Lys-Ala-Gln-Gly- (SEQ ID NO: 2) Gly-Ser-Asn  (3)PyroGlu-Ala-Lys-Ser-Gln-Gly- (SEQ ID NO: 3) Gly-Ser-Gln  (4)PyroGlu-Ala-Lys-Ser-Gln-Gly- (SEQ ID NO: 4) Gly-Ser-β-Ala-NH2  (5)PyroGlu-Ala-Lys-Ser-Gln-Gly- (SEQ ID NO: 5) Gly-Ser-D-Asn  (6)PyroGlu-Ala-Lys-Ser-Gln-Gly- (SEQ ID NO: 6) Gly-Ser-Asn-NH2  (7)PyroGlu-Ala-Lys-Ser-Asn-Gly- (SEQ ID NO: 7) Gly-Ser-Asn  (8)PyroGlu-Ala-Lys-Ser-Nva-Gly (SEQ ID NO: 8) Gly-Ser-Asn  (9)PyroGlu-Ala-Lys-Ser-Gln-Gly (SEQ ID NO: 9) Gly-Ala-Asp (10)Gln-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 10) Ser-Asp (11)Gln-Ala-Lys-Ala-Gln-Gly-Gly- (SEQ ID NO: 11) Ser-Asn (12)Gln-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 12) Ser-Gln (13)Gln-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 13) Ser-β-Ala-NH2 (14)Gln-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 14) Ser-D-Asn (15)Gln-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 15) Ser-Asn-NH2 (16)Gln-Ala-Lys-Ser-Asn-Gly-Gly- (SEQ ID NO: 16) Ser-Asn (17)Gln-Ala-Lys-Ser-Nva-Gly-Gly- (SEQ ID NO: 17) Ser-Asn (18)Gln-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 18) Ala-Asp (19)Glu-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 19) Ser-Asp (20)Glu-Ala-Lys-Ala-Gln-Gly-Gly- (SEQ ID NO: 20) Ser-Asn (21)Glu-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 21) Ser-Gln (22)Glu-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 22) Ser-β-Ala-NH2 (23)Glu-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 23) Ser-D-Asn (24)Glu-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 24) Ser-Asn-NH2 (25)Glu-Ala-Lys-Ser-Asn-Gly-Gly- (SEQ ID NO: 25) Ser-Asn (26)Glu-Ala-Lys-Ser-Nva-Gly-Gly- (SEQ ID NO: 26) Ser-Asn (27)Glu-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 27) Ala-Asp (28)Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 28) Ser-Asp (29)Ala-Lys-Ala-Gln-Gly-Gly- (SEQ ID NO: 29) Ser-Asn (30)Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 30) Ser-Gln (31)Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 31) Ser-β-Ala-NH2 (32)Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 32) Ser-D-Asn (33)Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 33) Ser-Asn-NH2 (34)Ala-Lys-Ser-Asn-Gly-Gly- (SEQ ID NO: 34) Ser-Asn (35)Ala-Lys-Ser-Nva-Gly-Gly- (SEQ ID NO: 35) Ser-Asn (36)Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 36) Ala-Asp (37)Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 37) Ser-Asp (38) Lys-Ala-Gln-Gly-Gly-(SEQ ID NO: 38) Ser-Asn (39) Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 39)Ser-Gln (40) Lys-Ser-Gln-Gly-Gly-Ser-β- (SEQ ID NO: 40) Ala-NH2 (41)Lys-Ser-Gln-Gly-Gly-Ser- (SEQ ID NO: 41) D-Asn (42)Lys-Ser-Gln-Gly-Gly-Ser- (SEQ ID NO: 42) Asn-NH2 (43)Lys-Ser-Asn-Gly-Gly- (SEQ ID NO: 43) Ser-Asn (44) Lys-Ser-Nva-Gly-Gly-(SEQ ID NO: 44) Ser-Asn (45) Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 45)Ala-AspMore specifically, the invention envisages protection of utilization ofthe following peptides:

(19) Glu-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 19) Ser-Asp  (1)PyroGlu-Ala-Lys-Ser-Gln-Gly- (SEQ ID NO: 1) Gly-Ser-Asp (10)Gln-Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 10) Ser-Asp (28)Ala-Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 28) Ser-Asp (37)Lys-Ser-Gln-Gly-Gly- (SEQ ID NO: 37) Ser-Asp

These peptides have been studied in two models of inflammation andhyperalgesia in the rat, which were induced either by intraplantarinjections (localized) or by intraperitoneal injections (systemic) ofendotoxins. Pretreatment using these peptides dose—dependently abolishesboth mechanical hyperalgesia and thermal hyperalgesia. In addition,pretreatment significantly reduces hyperproduction of IL-1β, IL-6, TNFαand NGF due to an intraplantar injection of endotoxin. In the case ofintraperitoneal injection, which has the effect of a state comparable toseptic shock (pain, fever, somnolence and anorexia), pretreatmentprevents hyperalgesia and maintains the body at normal temperature.

Finally, these peptides have analgesic effects that are identical orsuperior to those of the other anti-inflammatory drugs, while notinducing any obvious change in physiological or behavioral parametersfor all of the doses used.

Thus, these peptides have analgesic and/or anti-inflammatory properties.

The analgesic properties of these peptides extend also to neurogenicpain (or neuropathic pain). Several models have been used to testneurogenic pain of different etiologies: two animal models ofmono-neuropathy, another pain model of the somatic or visceral type.These models have made it possible to confirm a significant reduction ofthe mechanical allodynia and, at times, thermal allodynia. Theneuropathic manifestations are inhibited, while behavior associated withpain induced by the injection of irritating substances (capsaicin) isreduced. In all cases, these peptides have inhibitory effects that aregreater or equal to those induced by other treatments utilized in casesof neurogenic pain.

Considering their properties, the utilization of these peptides is moreparticularly recommended in the treatment of migraine, sciatica,neuropathy and acute or chronic pain of inflammatory origin.

For optimum efficacy, the doses administered must be between 0.01 and 1mg/kg, said administration being done by any variety of routes,including the parenteral, transcutaneous or nasal.

Other features and advantages of the invention will be better understoodin view of the following examples, while referring to the figuresrepresenting, respectively:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the dose-dependent reduction of hyperalgesia inducedby an injection of endotoxins;

FIG. 2 represents a comparative study of the anti-hyperalgesic effectsof the peptides according to the invention compared with steroids,non-steroidal anti-inflammatories and analgesic tri-peptides;

FIG. 3 represents the anti-inflammatory effects of pretreatment with thepeptides according to the invention, by reduction of the concentrationsof pro-inflammatory cytokines and NGF;

FIG. 4 represents the effects of pretreatment relative to pain (A and B)and to fever (c) induced by systemic injection of endotoxins insimulation of septic shock;

FIG. 5 represents the inhibition of allodynia (abnormal pain induced byinoffensive stimuli) and hyperpathy (exaggerated reaction to anociceptive stimulation of moderate intensity) in rats with a CCI(chronic compression of the sciatic nerve) mono-neuropathy, pretreatedwith the peptides according to the invention;

FIG. 6 represents the inhibition of allodynia and hyperpathy in ratswith an SNI (spared nerve pain of a enervated paw) mononeuropathy,pretreated with the peptides according to the invention;

FIG. 7 represents a dose study of the peptides according to theinvention in rats having the two aforementioned types of neuropathy;

FIG. 8 represents a dose study of the peptides according to theinvention in rats having hyperalgesia induced by the injection ofcapsaicin (somatic neurogenic pain);

FIG. 9 represents the attenuation by the peptides according to theinvention of the visceral pain induced by an intraperitoneal injectionof capsaicin (visceral spasmodic neurogenic pain);

FIG. 10 represents the comparison of the different drugs for neuropathy,and

FIG. 11 represents the results of daily treatment using TAP.

EXAMPLES Example 1 Analgesic and Anti-Inflammatory Properties of thePeptides According to the Invention

1. Material and Methods:

The experiments were conducted using adult, male Sprague-Dawley ratsweighing between 200 and 250 g. The animals were reared under optimumconditions of light and temperature (12 h light and shadow cycle; 22±3°C.). Food and water were dispensed ad libitum. All of the experimentswere conducted in compliance with the ethical directives relative topain experiments done on animals in the conscious state (Zimmermann M.,1983, Ethical guidelines for investigations of experimental pain inconscious animals, Pain, 16:109-110) and they were approved by theInstitutional Review Board for animal care.

Behavioral Procedures

Thermal and mechanical pain tests were done over a period of 3consecutive days prior to the injections, in order to establish abaseline.

The mechanical nociceptive threshold was determined by the mechanicalpaw pressure test (PP), while that of the thermal nociceptive thresholdwas determined using the hot-plate (HP), the paw immersion in hot water(PI) and the tail flick (TF) tests.

The PP test is done by applying a constant pressure of 0.20 g/cm²alternatingly to the left and right hind paws in an interval of 5minutes between 2 consecutive pressure applications. The pressure isstopped when that animal exhibits a typical reaction characterized by avigorous flexion reflex.

In the HP test, the animals are placed individually on a hot plate(52.5° C.±0.3° C.). The pain threshold is measured by the latency periodlapsed between the moment, at which the animal is placed on the hotplate and the first sign of pain, indicated by the fact that the animallicks the paw or jumps.

In the PI test, the hind paws are wetted alternately in distilled waterat 48° C. and the latency time lapsed until the first sign of withdrawalof the paw is noted.

In the TF test, the tail of each animal is immersed in distilled waterat 50.5° C. The latency time taken by the animal to withdraw it isnoted. The results are based on 3 consecutive trials done in 5 minuteintervals.

Administration of the Drugs

Inflammatory hyperalgesia was done using two animal models: one forlocalized inflammation; the other for systemic inflammation.

In the so-called localized model, the rats received an intraplantarinjection of a solution (1.25 μg in 50 μl of 9‰ physiological salinesolution) of endotoxin (Salmonella typhasa lipopolysaccharides, Sigma)into one of the hind paws, which induces both thermal and mechanicalhyperalgesia restricted to the paw having received the injection.

In the second model, the rats received an intraperitoneal injection ofendotoxin (25 μg in 100 μl of physiological saline solution).

Various treatments are then possible:

Protocol 1(a): different groups of rats (n=5 in each group) were treatedwith the analogue peptide (TAP) Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asp (SEQID NO: 19) (synthesized by Quantum Biotechnologies, Inc., Canada), inthe following fashion:

-   -   either, they received one intraperitoneal injection (25 μg in 50        μl of physiological saline solution) of this peptide;    -   or, they were pretreated with different doses of this peptide        (1.5 and 25 μg in 50 μl of sodium chloride) by intraperitoneal        injection, 30 minutes prior to injection of endotoxins (ET)        (1.25 μg in 50 μl of physiological saline solution by        intraplantar injection).

Protocol 1(b): other experiments looked at comparing the efficacy of theanalog peptides with that of steroids, NSAIDs and peptides known fortheir antagonism of the hyperalgesia induced by IL-1β and theprostaglandins.

One group of rats was pretreated with intraperitoneal injections ofLys-D-Pro-Thr (10 mg/kg) in 100 μl of physiological saline solution, 30minutes prior to injection of TAP. This tripeptide is known for itsantagonism of the hyperalgesia induced by IL-1β only.

In the second group, the rats received intraperitoneal injections of theLys-D-Pro-Val tripeptide (10 mg/kg) in 100 μl of saline solution, 30minutes prior to the injection of TAP. Lys-D-Pro-Val is an antagonist ofthe hyperalgesia induced by IL-1β and PEG₂.

The doses utilized for these tripeptides are those described in thearticle by Safieh-Garabedian (Safieh-Garabedian B., Kanaan S. A., HaddadJ. J., Abou Jaoude P., Jabbur S. J., Saade N. E. 1997, Involvement ofinterleukin-1β, nerve growth factor and prostaglandin-E2 in endotoxininduced localized inflammatory hyperalgesia. Brit. J. Pharmacol. 121:1619-1626).

A third and a fourth group were treated with dexamethasone andindomethacin.

Dexamethasone phosphate dissolved in a 9‰ sodium solution was injected,at a concentration of 200 μg/kg, just before and 3 hours after theinjection of ET.

Indomethacin was prepared by dissolving the indomethacin lactose in asaline buffer solution (pH 7.4) and was injected at a concentration of 4mg/kg, just before and 3 hours after the injection of ET.

All of the tests of the aforementioned experiments were done 9 hoursafter injection of the ET. This coincides with the hyperalgesia peak inthis model. The injection of saline solution (50-100 μl intraplantar)did not demonstrate significant alteration of the pain threshold.

Protocol 2(a): One group of rats received one intraperitoneal injectionof ET (50 μg), while the other group was pretreated with the proteinanalogue of thymulin (TAP) (25 μg, intraperitoneal injection), 30minutes before the injection of ET.

The TF and PP tests were then done at 1 hour, 3 hours and 6 hours afterthe injection of ET.

Protocol 2(b): Different groups of rats (n=5 in each group) were treatedin the following fashion:

-   -   either they received one intraperitoneal injection of 50 μg of        ET,    -   or they were pretreated with the TAP peptide (25 μg,        intraperitoneal injection), 30 minutes before an injection of        endotoxin.

Rectal temperature was measured at 1 hour, 3 hours and 6 hours.

A control group received an intraperitoneal injection of TAP peptide (25μg dissolved in 100 μl of saline solution).

Cytokines and Nerve Growth Factor (NGF)

These experiments required tissue sampling. The animals were sacrificedby anesthesia (sodium penthiobarbital, 50 mg/kg) and the skin of thehind paws is sampled either at 1 h (for the determination of the TNFα)or at 4 h (for the determination of IL-1β, IL-6 and NGF) after theendotoxin injection.

These tissue samples are weighed and then quick frozen and stored at−70° C. with a view of proceeding with the evaluation of IL-1β, TNFα,IL-6 and NGF.

In another series of experiments, the tissues are taken as describedabove using different groups of rats; the one group being pretreatedwith TAP 30 minutes before injection of ET and the other group havingbeen injected with TAP only.

The tissues are homogenized in a phosphate buffer solution (PBS,pH=7.4), containing 0.4 M of NaCl, 0.05% of Tween-20®, 0.5% bovine serumalbumin (BSA), 0.1 mM of phenylmethylsulfonyl fluoride, 0.1 mM ofbenzethonium chloride, 10 mM of EDTA and 20 KI/mL of aprotinin.

The mixture is then centrifuged at 1,200 g for 60 minutes at 4° C. Thecytokines and the NFG contained in the supernatant were measured usingELIZA assays.

The NGF is measured with the aid of an immunological kit (Promega) byfollowing the recommended instructions of the manufacturer.

Measurement of the IL-1β, TNF-α and IL-6 was done in accordance with theprotocols described by Safieh-Garabedian (Safieh-Garabedian B., DardenneM., Kanaan S. A., Atweh S. F., Jabbur S. J., Saadé N. E. 2000. The roleof cytokines and prostaglandin-E2 in thymulin induced hyperalgesia.Neuropharmacology 39:1653-1661).

Statistical Analysis and Treatment of the Data

A pain threshold value for the different nociceptive tests was definedfor each group of animals. The data obtained for each drug tested werecompared either to the control established before the injection or withtwo types of controls: a series of animals receiving one injection of ETand another series of animals receiving one injection of 9‰ sodiumchloride solution.

For the evaluation of the cytokine and NFG levels, the values obtainedusing the animals injected with ET alone, drug alone or ET plus drugwere compared to the values obtained using the groups of control animalsinjected with a 9‰ sodium chloride solution.

The significance of the standard deviation was established using ANOVAfollowed by a Bonferroni test.

2. Results

Effect of the TAP Peptide on Inflammatory Hyperalgesia Induced byIntraplantar Injection of ET.

The intraplantar injection of ET (1.25 μg in 50 μl of saline solution)into the hind paw of rats induced a significant reduction of thenociceptive thresholds measured at 9 h (peak of hyperalgesia) accordingto the PP test (0.87±0.09 s compared with 2.06±0.06 s for the controlsaline solution, P<0.001) for mechanical hyperalgesia and according tothe PI test (1.25±0.04 s compared with 1.97±0.05 s for the controlsaline solution, P<0.001), HP (6.0±0.18 compared with 9.24±0.16 s forthe control saline solution, P<0.001) and TF (2.40±0.06 s compared with3.19±0.08 s for the control saline solution, P<0.001) for thermalhyperalgesia. Treatment using the TAP peptide (1.5 and 25 μg) reduced indose—dependent fashion the hyperalgesia induced by the injection of ET(FIG. 1). With the 25 μg dose of TAP, the latency for triggering thevarious responses were from 2.05±0.07 s, 1.94±0.005 s, 9.12±0.7 s and3.22±0.09 s for the PP, PI, HP and TF tests, respectively (p>0.05 forall of the values in comparison with the baseline or with the valuesobtained using saline solution). Intraperitoneal injection of TAP alone(25 μg in 50 μl of saline solution) did not result in significant changein the latency time in the different pain tests.

Comparison of the Efficacy of the Tap Peptide with Other Medicines andAnalogues

When the effect of TAP on hyperalgesia induced by an intraplantarinjection of ET (1.25 μg) is compared with the effects obtained using asteroid, an NSAID and the Lys-D-Pro-Val et Lys-D-Pro-Thr peptides, theresults obtained demonstrate that TAP is a much more efficaciousanalgesic agent than the Lys-D-Pro-Val et Lys-D-Pro-Thr peptides (FIG.2). TAP has effects similar to those of indomethacin and dexamethasonebut at much lower concentrations (FIG. 2).

Effect of TAP on the Cytokines

Injection of endotoxins into the hind paw of rats induced a significantincrease (p<0.001) of the pro-inflammatory cytokine and NGFconcentrations and in comparison with the rats injected with salinesolution or with the paws of the same rats not having received anyproduct. One hour after injection of ET, the concentration of TNF-α was345.0±61.0 pg/paw compared with 100.0±8.00 pg/paw after injection ofcontrol saline solution. Three hours after injection of ET, theconcentration of IL-1β was 2,850.6±255.4 pg/paw compared with 400.0±45.0pg/paw after injection of the control saline solution; the concentrationof IL-6 was 2,831.0±285.0 pg/paw compared with 250.0±50.0 pg/paw for thecontrol saline solution and the NGF concentration was 23.0±1.73 ng/pawcompared with 9.11±1.6 ng/paw for the control saline solution.

Previous treatment with TAP nullified the increase of the TNF-αconcentration and significantly reduced the IL-1β concentrations (from2850.6±255.4 to 1686.0±266.0 pg/paw, P<0.01), IL-6 (from 2831±285 to1158±197.0 pg/paw, p<0.001) and NGF (from 23.0±1.73 to 16.73±2.70ng/paw, p<0.001) (FIGS. 3A, B, C and D). Injection of TAP (25 μg) incontrol animals did not induce significant change of the cytokine or NGFconcentrations, as shown in FIG. 3.

Effect of TAP on Systemic Inflammatory Hyperalgesia Induced by ET.

Injections of ET (50 μg, i.p.) caused a significant reduction of thenociceptive thresholds at one hour according to the PP test (1.13±0.06compared with 2.03±0.04 s for the controls, p<0.001) and according tothe TF test (2.27±0.06 compared with 3.08±0.04 s for the controls,p<0.001). Hyperalgesia remained observable for six to nine hours afterthe injection (FIGS. 4A and 4B). Previous treatment by intraperitonealinjection of TAP (25 μg) thirty minutes before systemic administrationof ET abolished the mechanical hyperalgesia (FIG. 4A) and the thermalhyperalgesia (FIG. 4B) produced by the ET.

Endotoxin is a known pyrogen and its injection induced an significantincrease in body temperature at one hour (38.43±0.028° C. compared with37.65±0.16° C. for the controls). The temperature then remainedsignificantly elevated at six hours (FIG. 4C). Previous treatment withTAP in intraperitoneal injection (25 μg) nullified the process ofincrease of body temperature induced by ET and the values were notsignificantly different from those of the controls (FIG. 4C).

Example 2 Analgesic Properties of a Peptide According to the Invention(TAP) Relative to Pain of Neurogenic Etiology

1. Material and Methods:

Adult male Sprague-Dawley rats (250-300 g) were used for these trials.Over the period of the experiments, the rats were placed under standardconditions (4 to 5 individuals to a cage, 12 hour day/night cycle, 22.2°C.), with free access to water and food. The necessary surgicalprocedures were done under deep anesthesia using ketamine (KETALAR®,40-50 mg/kg, intraperitoneal injection), preceded by pre-anesthesiausing chlorpromazine (8 mg/kg, idem) and atropine (0.05 mg/kg, idem).

This study was based on two experimental protocols for the induction ofpain of neurogenic etiology. The first protocol utilized two animalmodels of mononeuropathy. The second protocol was based on the injectionof capsaicin, a substance known to activate the specific afferent fibergroups implicated in nociceptive signaling.

a) Protocol I: Animal Models of Mononeuropathy

Induction of Mononeuropathy:

Mononeuropathy was induced in different groups of rats (n=6 rats in eachgroup) according to the CCI (chronic constriction injury, chronic nerveconstriction, Bennet G. J. et Xie Y. K., 1988, A peripheralmononeuropathy in rat that produces disorders of pain sensation likethose seen in man, Pain, 33:87-107) or according to the SNI (sparednerve injury, spared nerve of the enervated paw, Decosterd I. et WoolfC. J., 2000, Spared nerve injury: an animal model of persistentperipheral neuropathic pain, Pain, 87:149-158). The sciatic nerve wasexposed after dissection of the posterior hip and by incision throughthe skin and the fatty layer covering the popliteal fossa. For the CCImodel, four loose ligatures (chromed catgut 4.0) were placed on theproximal side of the sciatic trifurcation. For the SNI model, theexternal popliteal sciatic nerve and the internal popliteal sciaticnerve were isolated, tightly ligatured and sectioned, while the suralnerve innervating the lateral aspect of the paw was left intact.

Behavioral Tests:

The rats were placed in individual compartments of one cage, whose floorwas made of a metal grating allowing access to the heel pad and to thelateral aspect of the paw with wires.

For mechanical allodynia, the plantar surface of the hind paws (lateralsurface for the SNI model and the median plantar surface for the CCI)was contacted with the von Frey wires (VFF 4.31 and 5.07, Stoelting Co.,USA), which corresponded to the forces of 2.041 and 11.749 g (18.5 and106.7 mN), respectively. The flexion forces of these wires were shown tobe insufficient for causing nociceptive withdrawal reflexes in normalanimals. The number of paw retractions induced by 10 trials wasestablished for each rat on each hind paw prior to (baseline) and afterinduction of the mono-neuropathy. In normal rats, the low caliber wires(VFF 4.31) and higher caliber (VFF 5.07) induced an average 1.3±0.2 and2.7±0.3 responses/10 trials, respectively.

After induction of neuropathy, the two filaments produced more than 5responses/10 trials.

The method described by Choi et al. (1994, Behavioral signs of ongoingpain and cold allodynia in a rat model of neuropathic pain, Pain,59:369-376) was used for evaluation of cold allodynia. It consists ofapplying several drops (approximately 50 μl) of an acetone solution onthe paw and measuring the duration of the retraction reaction. One halfsecond and 20 seconds are the values chosen arbitrarily for the minimumand maximum threshold, respectively.

The duration (D) of retraction of the paw (RP) in response to anociceptive ray of radiant heat oriented towards the plantar surface wasestablished in the normal rat. Increasing the DRP after induction of themononeuropathy was considered to be an indication of hyperalgesia. Eachrat underwent two RP tests per session a minimum of every five minutes.

The neuropathic manifestations were at maximum seven to ten days afterinduction of the neuropathy. The effects of the injections of TAP weretested during this period, which corresponds to the peak of theneuropathy.

b) Protocol II: Chemical Irritation of the Nociceptive Afferents

This protocol was based on the established properties of capsaicin,which selectively irritates a specific group of afferent fibers (called“capsaicin sensitive primary afferents” or CSPA) known to produce aneurogenic inflammation and for transmitting the nociceptive information(for a review, see Szolcsany J., 1996, Neurologic inflammation:reevaluation of axon reflex theory, In: Geppetti P. and Holzer P.(Eds.), Neurogenic Inflammation, pp. 33-42. CRC Press, Boca Raton).

Our group applied two methods: intra-plantar (i. pl.) andintraperitoneal (i.p.) injection in low quantities of capsaicin in orderto provoke reversible somatic and visceral pain, respectively.

Intraplantar Injection of Capsaicin

Injection of capsaicin (10 μg in 50 μl of a 10% solution of Tween 20 inolive oil) produced hyperalgesia, whose peak was at three to six hoursafter injection and which disappeared at the end of 24 hours. Mechanicalhyperalgesia was evaluated using the paw pressure test (PP), whichconsists in applying a constant pressure of 0.2 kg/cm on the dorsal partof the hind paw. The time lapsed between the application of the pressureand the nociceptive reflex of retraction of the paw was considered asthe latency time (or threshold) of mechanical nociceptive. Thermalhyperalgesia was evaluated using the hot-plate (HP) test and the pawimmersion (PI) test.

The HP and PI tests were done as in Example 1, the hot-plate having atemperature 52.5±0.3° C. and the hot water container at 48±0.3° C.

The PP and PI tests were done one after the other on the two hind pawsobserving an interval of five minutes between two consecutive tests.

The rats were sent to the laboratory one week prior to injection so thatthey would become accustomed to the environment and the tests were doneon the rats over two or three days in order to obtain the baseline valuefor each test before any treatment (detailed description in Kanaan S. A.et al., 1996, Endotoxin-induced local inflammation and hyperalgesia inrats and mice: a new model for inflammatory pain, Pain, 66:373-379).

Intraperitoneal Injection of Capsaicin

This test consisted of injecting (i.p.) 20 μg of capsaicin in 100 μl ofa 10% solution Tween 20 in olive oil in some rats and observing thebehavior induced by these injections. A 4-level behavioral scale wasdesigned according to the method described by Giesler G J et al., (1976,Inhibition of visceral pain by electrical stimulation of theperiaqueductal gray matter, Pain 2:43-48).

The levels were defined as follows: 0=normal behavior; 1=slightcontraction of the abdominal muscles; 2=contraction of one single sideand luxation of the haunch dropping; 3=significant contraction of theabdominal muscles and extension of the two rear paws.

For the evaluation of the animals' behavior, each rat was placed intransparent cage placed over a mirror inclined at 450 for optimumobservation.

Normal and nociceptive behaviors were noted by one observer using apolygraph and the time corresponding to each level over a period of 30minutes was accounted for regarding the polygraph recordings by anotherobserver. None of the two observers knew the injection administered northe expected effects.

Medicines Injected

The TAP peptide used is the same as that used in Example 1.

The capsaicin (8-methyl-N, vanillyl-nonanamide, Sigma #M1022) wasdissolved in a 10% solution of Tween 20 in olive oil and administeredi.p. or i.pl. at the appropriate concentration.

Analysis of the Data

During the experiments done on rats suffering from neuropathy, allodyniaand hyperpathy were evaluated in each group of animals with reference tothe baseline condition established prior to induction of mononeuropathy.For example, the average of the number of paw withdrawal induced by eachVFF was calculated for all of the rats of each group and the variationof this mean has been determined after treatment with TAP at differenttime periods after the injection. The same method has been followed forallodynia in the code and the hypertrophy relative to heat.

In the course of the experiments involving the injection i.p. ofcapsaicin, the mean of the measurements done using each pain test (PP,HP or PI) was calculated for each group of rats prior to the injectionof the capsaicin (baseline condition) and at different time intervalsafter injection (3, 6, 9 and 24 hours). TAP was injected (i.p.) thirtyminutes before the capsaicin and the latency times of the different paintests were measured at the same intervals of time after injection of thecapsaicin.

For the injection i.p. of the capsaicin, the total time corresponding toeach behavioral level was measured for the animals having received thecapsaicin alone or the capsaicin after the TAP. The variation of theneuropathic manifestations or of the pain tests after the treatmentswere evaluated by ANOVA then by Bonfrerroni tests done post hoc. Thevariations of the pain scores with or without treatment were evaluatedby the Student bilateral test by assuming a significance level of 5%(p<0.05).

2. Results

Effects of the Injection of Tap on Neuropathic Manifestations

Injection of TAP (5 μg in 100 μl, i.p.) in rats of two groups (n=6 ratsper group) subjected to neuropathy induced by CCI or SNI resulted in asignificant attenuation of all of the neuropathic manifestations (FIGS.5 and 6). This effect was maximum for mechanical allodynia at two hoursafter injection. It was maximum for cold allodynia at 75 minutes afterinjection and the hyperpathy in the CCI model and for all of theneuropathic manifestations in the SNI model. However, the cold allodyniawas only moderately reduced by TAP in the SNI model. Reversibility ofthe effects of TAP was observed three to four hours after injection.

The effects of doses of TAP of 1 and 25 μg were evaluated in othergroups of rats by following the two models. FIG. 7 shows that theattenuation of the neuropathic manifestations was maximal using a doseof 5 μg/rat.

Daily treatment with TAP (1 μg in 100 μL) over 5 consecutive daysproduces a progressive reduction of the manifestations of the allodyniaas well as a potentiation of the effects of each injection. The mostobvious proof of this potentiation is the increased inhibition of thecold allodynia that was little changed by a single injection of TAP (cf.FIG. 11).

The effects of treatment with TAP on the manifestations of neuropathywere compared to those observed with injections of either meloxican (5mg/kg, i.p.) or morphine (4 mg/kg, i.p.) in two other groups of rats(n=6). Treatment with TAP induces a greater reduction of tactileallodynia and of thermal hyperalgesia that that observed with the twoother drugs and this and a much lower dose. FIG. 10 represents thiscomparison. Each drug is administered by injection to a different group(n=6) of rats subjected to a neuropathy induced by SNI. All of themeasurements were done at the peak of activity of each drug (45-60minutes after the injection). The controls corresponding to the measuresdone on the rats subjected to a neuropathy prior to any treatment.

Effects of Treatment with TAP on Hyperalgesia Induced by the Injectioni.pl. of Capsaicin

Intra-plantar injection of capsaicin (10 μg in 50 μl) resulted in asignificant reduction of the latency times (hyperalgesia) observed atthe time of the different pain tests. This reduction was maximum at theend of three to six hours and disappeared 24 hours after injection(Saade N. E., Massaad C. A., Ochoa-Chaar C. I., Atweh S. F.,Safieh-Garabedian B., Jabbur S. J. 2000. Possible contribution ofneuropeptides and histamine to the hyperalgesia induced by intraplantarinjection of capsaicin. Eur. J. Neurosci. Abst. (suppl 11, Vol. 12:123). Different groups of rats (n=5 rats in each group) were injectedeither with capsaicin or TAP (i.p.) then the capsaicin at the end ofthirty minutes at doses of 1.5 or 25 μg/rat. The previous treatment withTAP resulted in a dose-dependent attenuation of the hyperalgesia inducedby the capsaicin (FIG. 8). The more elevated dose, the injection of TAPinvolved a complete prevention of the hyperalgesia induced by thecapsaicin. Injection of a dose of 25 μg of TAP did not result insignificant modification of the latency times observed at the time ofthe different pain tests (FIG. 8, controls).

Effects of the Injection of TAP on Visceral Pain Induced by Capsaicin

Rats (n=6) having received an injection i.p. of capsaicin (20 μg in 100μl) had a response corresponding to level 0 over 0.2±0.3 minutes, level1 over 2.51±0.45 minutes, level 2 over 21.5±0.8 minutes and level 3 over11.84±0.43 minutes over a total observation period of 36 minutes (FIG.9).

In another group of rats (n=6), injection of TAP (50 μg in 200 μl/rat)prior to injection of capsaicin (20 μg, i.p.), the following scoresresulted: 0.82±0.18 minutes corresponding to level 0, 12.89±2.5 minutescorresponding to level 1, 15.62±0.9 minutes corresponding to level 2 and6.67±0.09 minutes corresponding to level 3. Accordingly, prior treatmentwith TAP resulted in a significant shift to the left of the nociceptionscores induced by capsaicin (FIG. 9).

Example 3 Utilization of TAP for Manufacturing an Injectable Solution

-   -   TAP: 0.05 mg    -   Sterile, pyrogen-free distilled water 1.0 ml.

Sterilization by filtration, packaging in ampoules, bottles or multipledose bottles.

Possible routes of administration are intraperitoneal, sub-cutaneous,intracerebral, intramuscular and intradermal injection.

1. A method for treating pain, wherein said method comprisesadministering to a patient in need of said pain treatment an effectiveamount of a composition comprising at least one immune-system-inactivethymulin peptide analogue, wherein said analogue does not contain zinc,and wherein said peptide analogue is Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asp(SEQ ID NO: 19) and wherein said composition is administered in anamount between 1 μg/kg and 10 mg/kg.
 2. The method according to claim 1,wherein said composition further comprises a medicine having analgesicproperties and/or anti-inflammatory properties.
 3. The method accordingto claim 1, wherein said pain is at selected from the group consistingof: a migraine, sciatica, neuropathy and inflammatory pain.
 4. Themethod according to claim 1, wherein said composition is administeredparenterally or nasally.